ABSTRACT The clinical use of pro-angiogenic growth factors could greatly impact the treatment of critical limb ischemia (CLI), a condition characterized by arterial blockages in the extremities. With CLI, 40% of patients are ineligible for available therapies and even with intervention, the 6-month risk of limb amputation is 25-40% with an annual mortality of 20%. In preclinical models of CLI, collateral blood vessel formation and perfusion restoration are observed in the ischemic limb following the administration of angiogenic growth factors. However, attempts at clinically translating these promising preclinical results, via the use of at-site or systemic injections of angiogenic growth factors, has remained a challenge. The delivery of growth factors via injection or using conventional scaffold-based approaches does not afford active control of the dose, timing, or spatial localization at the intended site of collateral vessel formation. Furthermore, no consensus exists regarding what range of these parameters, including what combination of growth factors, are required for effective therapeutic angiogenesis. Thus, there is an urgent need to develop a safe and effective delivery system for multiple angiogenic growth factors that recapitulates critical aspects of endogenous growth factor signaling and facilitates identification of these crucial parameters. Our long-term goal is to develop implantable biomaterials for the delivery of regenerative molecules, where delivery can be manipulated spatiotemporally in an externally-regulated, on-demand manner. The modulating mechanism is megahertz-range ultrasound, which is clinically translatable since it can be applied non-invasively, focused with sub-millimeter precision, and delivered in a spatiotemporally defined manner to sites deep within the body. The objective of this proposal is to develop an implantable scaffold where the released dose, sequence, and localization of two growth factors involved in angiogenesis - basic fibroblast growth factor (bFGF) and platelet derived growth factor-BB (PDGF- BB) - are non-invasively controlled. The scaffold, termed an acoustically-responsive scaffold (ARS), is doped with two ultrasound-sensitive emulsions that each contain a growth factor. The central hypothesis driving this project is that ultrasound can spatiotemporally pattern angiogenesis in and around an ARS by controlling the sequential release of bFGF and PDGF-BB. The rationale for the proposed research is that an ARS enables the study of how various doses and spatiotemporal gradients of bFGF and PDGF-BB affect the development of blood vessels, which can be used in the translation of therapeutic angiogenesis for the treatment of CLI. The hypothesis will be tested via three specific aims: 1) enhance selective release of growth factors from the ARS; 2) use an ARS to demonstrate the impact of spatiotemporally-generated gradients of bFGF on angiogenesis; and 3) demonstrate restoration of perfusion in a murine hind limb ischemia model using an ARS. Successful completion of the proposed research is significant since it will elucidate how microenvironmental factors ? such as growth factor doses, spatiotemporal profiles, and sequence ? affect angiogenesis.